Reagent combination for biosensor, biosensor and application thereof in cancer marker detection

Abstract

The present disclosure provides a biosensor reagent combination, a biosensor, and its application in cancer marker detection. The biosensor reagent combination includes a substrate material and platinum-coated gold nanorod probes. The substrate material includes NH2-MXene@AuNPs and biosynthetic quantum dots. The biosensor reagent combination, biosensor, and its application in cancer marker detection provided herein can effectively improve detection sensitivity, accuracy, and speed, while effectively reducing detection difficulty, and are of great significance for the in-depth application of biosensors in medical clinical diagnosis.

Description

Technical Field

[0001] The present application relates to the technical field of biosensors, and in particular to a reagent combination for a biosensor, a biosensor, and its application in the detection of cancer markers. Background Art

[0002] Biosensors are analytical devices used to detect analytes. Through a series of reactions, they convert properties such as the analyte's type and concentration into easily accessible quantitative data for subsequent analysis. These sensors have broad applications in biomedical research, drug synthesis and screening, environmental monitoring and protection, health quarantine, forensic identification, and biomarker detection. However, existing biosensors have limited sensitivity, struggling to achieve the low picogram per milliliter (pg / mL) sensitivity required for clinical applications. Therefore, developing more sensitive biosensors is crucial for the further application of sensors in multiple industries. Summary of the Invention

[0003] In view of this, the purpose of this application is to propose a reagent combination for a biosensor, a biosensor and its application in cancer marker detection, so as to solve the above technical problems.

[0004] In a first aspect of the present application, a reagent combination for a biosensor is provided, comprising a substrate material and a platinum-coated gold nanorod probe, wherein the substrate material comprises NH2-MXene@AuNPs and biosynthetic quantum dots.

[0005] In a second aspect of the present application, a biosensor is provided for detecting a target protein, comprising a sensing substrate and a platinum-coated gold nanorod probe, wherein the sensing substrate is prepared from NH2-MXene@AuNPs and biosynthetic quantum dots.

[0006] Furthermore, the biosensor is an electrochemical biosensor; the sensing substrate of the electrochemical biosensor is prepared by the following method:

[0007] NH2-MXene@AuNPs, biosynthetic quantum dots, a first antibody of a target protein, and bovine serum albumin are sequentially applied to a glassy carbon electrode to obtain a sensing substrate of the electrochemical biosensor.

[0008] Furthermore, the biosensor is a colorimetric biosensor; the sensing substrate of the colorimetric biosensor is prepared by the following method:

[0009] NH2-MXene@AuNPs, biosynthetic quantum dots, the first antibody of the target protein, and bovine serum albumin are sequentially added dropwise to the well plate to obtain the sensing substrate of the colorimetric biosensor.

[0010] Furthermore, the platinum-coated gold nanorod probe is prepared by the following method:

[0011] Gold nanorods were mixed with CTAB solution, AgNO3 solution and ascorbic acid solution, stirred, and then HCl and H2PtCl6 solution were added to react to obtain platinum-coated gold nanorods;

[0012] Bovine serum albumin is added to the platinum-coated gold nanorod solution for incubation, and after the incubation is completed, a second antibody of the target protein is added for reaction to obtain the platinum-coated gold nanorod probe.

[0013] Furthermore, the gold nanorods are prepared by the following method:

[0014] Mixing the HAuCl4 solution with the CTAB solution to obtain a first mixed solution;

[0015] Adding NaBH4 solution to the first mixed solution, stirring and allowing to stand to obtain a seed solution;

[0016] Completely dissolving CTAB and NaOL in deionized water to obtain a second mixed solution; sequentially adding AgNO3 solution, HAuCl4 solution, and acrylic acid solution to the second mixed solution to obtain a growth solution;

[0017] The seed solution is added to the growth solution to react and generate the gold nanorods.

[0018] Furthermore, the NH2-MXene@AuNPs were prepared by the following method:

[0019] Ti2CTx-Mxene was added to an ethanol solution and subjected to ultrasonic treatment to obtain a Ti2CTx-Mxene-ethanol solution;

[0020] APTES solution was added to Ti2CTx-MXene-ethanol solution to obtain NH2-MXene;

[0021] Ultrasonic mixing of NH2-MXene and gold nanoparticles to obtain the NH2-MXene@AuNPs;

[0022] Wherein, the gold nanoparticles are prepared by the following method:

[0023] trisodium citrate was added to deionized water, heated to boiling, and then HAuCl4 solution was added to obtain a third mixed solution;

[0024] The trisodium citrate solution and the HAuCl4 solution were added to the third mixed solution in batches for multiple times to obtain the gold nanoparticles.

[0025] Furthermore, the biosynthetic quantum dots are prepared by the following method:

[0026] Staphylococcus aureus was activated in NB liquid culture medium, and then a portion of the bacterial solution was transferred to LB liquid culture medium for culture. CdCl2 and sterile water were added to the LB liquid culture medium and incubated to synthesize the biosynthetic quantum dots.

[0027] Furthermore, the target proteins include metaphase protein, alpha-fetoprotein, carcinoembryonic antigen and prostate specific antigen.

[0028] The third aspect of the present application provides an application of a biosensor in the detection of cancer markers, wherein the concentration of metaphase protein is detected using a biosensor according to any one of the second aspects.

[0029] As can be seen from the above, the present application provides a reagent combination for a biosensor, a biosensor and its application in the detection of cancer markers, including NH2-MXene@AuNPs, biosynthetic quantum dots and platinum-coated gold nanorod probes. NH2-MXene@AuNPs has good electrical conductivity, so it can ensure the electrical conductivity of the sensing surface, and the large number of amino groups introduced can promote the connection with BQDs. BQDs can be effectively fixed on the NH2-MXene@AuNPs material through their own carboxyl groups. Since BQDs are obtained by biosynthesis and have protein A, when used for the detection of target proteins, they can be accurately connected to the Fc fragment of the first antibody of the target protein, thereby playing a role in directional fixation and efficient recognition of the first antibody. Platinum-coated gold nanorod probes have excellent electrochemical conductivity and unique catalytic properties for H₂O₂ and TMB (3,3',5,5'-tetramethylbenzidine). Whether used in electrochemical or colorimetric biosensors, they can amplify signals, effectively reducing false positive and false negative results during testing, thereby improving detection sensitivity and accuracy. The disclosed reagent combination for biosensors, biosensors, and their application in cancer marker detection can effectively improve detection sensitivity, accuracy, and speed, while effectively reducing detection difficulty, and are of great significance for the in-depth application of biosensors in medical clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in this application or related technologies, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are merely embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0031] Figure 1Ti2CT x -Mxene, NH2-MXene, AuNPs, NH2-MXene morphology characterization diagram, where A is Ti2CT x -MXene, B is the SEM image of NH2-MXene, C is the TEM image of AuNPs, and D is the TEM image of NH2-MXene@AuNPs.

[0032] Figure 2 This is a magnified TEM image of a single spherical gold nanoparticle.

[0033] Figure 3 The morphology characterization, size and element distribution diagram of BQDs, where A is the laser scanning confocal image of BQDs, B is the TEM image of BQDs, C is the size distribution diagram of BQDs (the horizontal axis is the size), and D is the elemental composition distribution diagram of BQDs (the horizontal axis is the element, and the vertical axis is the atomic ratio).

[0034] Figure 4 The morphology characterization diagrams of AuNR and Au@Pt NRs, where A and B are TEM images of AuNR at different magnifications, and C and D are TEM images of Au@Pt NRs at different magnifications.

[0035] Figure 5 This is the EDX spectrum of Au@Pt NRs (the horizontal axis is the radiation energy, and the vertical axis is the signal intensity).

[0036] Figure 6Schematic diagram of the conductive properties of GCE, NH2-MXene / GCE, NH2-MXene@AuNPs / GCE, BQDs / NH2-MXene@AuNPs / GCE, and MF-Probe, as well as the conductive properties of each stage in the manufacturing process of the electrochemical biosensor; A is the CV curve of GCE, NH2-MXene / GCE, and NH2-MXene@AuNPs / GCE, where a is the curve of GCE, b is the curve of NH2-MXene / GCE, and c is the curve of NH2-MXene@AuNPs / GCE, with the horizontal axis being the voltage and the vertical axis being the current; B is the CV curve of NH2-MXene@AuNPs / GCE before and after antibody incubation. DPV curves of Ps / GCE and BQDs / NH2-MXene@AuNPs / GCE, in which a is the DPV curve of BQDs / NH2-MXene@AuNPs / GCE after antibody incubation, b is the DPV curve of BQDs / NH2-MXene@AuNPs / GCE before antibody incubation, c is the DPV curve of NH2-MXene@AuNPs / GCE after antibody incubation, d is the DPV curve of NH2-MXene@AuNPs / GCE before antibody incubation, the horizontal axis is voltage, and the vertical axis is current; C is the DPV curve of MF-Probe, in which the horizontal axis is voltage, the vertical axis is current, Without MF-probe is the result without MF-probe, and With MF-probe is the result with MF-probe; D is the DPV curve of NH2-MXene@AuNPs and Au@Pt Schematic diagram of the catalytic ability of NRs for TMB, where the horizontal axis is wavelength and the vertical axis is absorbance, and Control is the curve of the control group; E is the EIS curve of the electrode at each stage in the preparation process of the electrochemical biosensor; F is the DPV curve of the electrode at each stage in the preparation process of the electrochemical biosensor, where the horizontal axis is voltage and the vertical axis is current; among them, in Figures E and F, a~g are the curves of GCE, NH2-MXene@AuNPs / GCE, BQDs / NH2-MXene@AuNPs / GCE, Ab1 / BQDs / NH2-MXene@AuNPs / GCE, BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE, MDK / BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE, and MF-Probe / MDK / BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE, respectively.

[0037] Figure 7Schematic diagram of the results of MDK detection by the biosensor; A is the DPV curve of the electrochemical biosensor detecting different concentrations of MDK, in which a~i are the curves of MDK with concentrations of 5fg / mL, 10fg / mL, 50fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, and 10ng / mL, respectively, with the horizontal axis being the voltage and the vertical axis being the current; B is the calibration curve of the electrochemical biosensor detecting different concentrations of MDK, in which the horizontal axis is the logarithm of the concentration and the vertical axis is the relative peak current; C is the UV-visible absorption spectrum of the colorimetric biosensor detecting different concentrations of MDK, in which a~i are 5pg / mL, 10pg / mL, 100fg / mL, 1ng / mL, 5ng / mL, respectively. L is the curve of MDK with concentrations of 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL, with the horizontal axis being the wavelength and the vertical axis being the absorbance; D is the calibration curve of the colorimetric biosensor for detecting different concentrations of MDK, with the horizontal axis being the logarithm of the concentration and the vertical axis being the absorbance; E is a schematic diagram of the color change of MDK with different concentrations detected by the colorimetric biosensor under sunlight, with the MDK concentrations of 5 pg / mL, 10 pg / mL, 100 fg / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL from left to right; F is the linear relationship between the RGB value of the color in Figure E and the MDK concentration, with the horizontal axis being the logarithm of the concentration and the vertical axis being the normalized RGB intensity.

[0038] Figure 8Figure 1 is a schematic diagram of the results of using the biosensor to detect the MDK concentration in the serum of healthy donors and thyroid cancer patients; A is a heat map of signal changes using electrochemical biosensors, colorimetric biosensors and ELISA equipment to detect the MDK concentration in serum, where Electrochemical Mode is the electrochemical mode, i.e., the detection result corresponding to the electrochemical biosensor, and the color bar label Current represents the current; Colorimetric Mode is the colorimetric mode, i.e., the detection result corresponding to the colorimetric biosensor, and the color bar label Absorbance represents the absorbance; the color bar label OD value in the ELISA heat map represents the OD value; B is a rectangular diagram of the MDK concentration in the serum of healthy donors and thyroid cancer patients (the horizontal axis Healthy represents the healthy donor, the horizontal axis Thyroid represents the MDK concentration in the serum of healthy donors and thyroid cancer patients) Cancer represents thyroid cancer patients, and the vertical axis represents MDK concentration); C is a scatter plot of MDK concentrations in the serum of healthy donors and thyroid cancer patients (***p<0.001, ****p<0.0001, t-test), where Normal represents normal healthy donors and Patients represents thyroid cancer patients; D is a receiver operating characteristic (ROC) curve for the clinical diagnosis of thyroid cancer using the biosensor, where AUC is the area under the curve. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of this application more clear, this application is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0040] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should have the usual meanings understood by people with ordinary skills in the field to which this application belongs. The "first", "second" and similar words used in the embodiments of the present application do not indicate any order, quantity or importance, but are only used to distinguish different components. "Include" or "comprise" and similar words mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0041] A biosensor is an analytical device used to detect analytes. It can convert the type, concentration and other properties of the analyte into quantitative data that is easily accepted by people through a series of reactions, facilitating subsequent analysis. It has a wide range of applications in many fields such as biomedical research, drug synthesis screening, environmental monitoring and protection, health quarantine, forensic identification, and biomarker detection.

[0042] Midkine (MDK), also known as midkine-related growth factor, is a soluble protein whose expression levels increase significantly during the development of cancer (including lung cancer and thyroid cancer). It has been shown to be detectable in urine and blood, and in recent years has been widely regarded as a promising cancer biomarker. Normally, MDK levels in the serum of healthy adults are very low (less than 600 pg / mL). However, as cancer progresses, MDK levels can exceed 1 ng / mL. Multiple reports indicate that MDK overexpression is also a key indicator of impaired prognosis in lung cancer patients. Currently, MDK, one of the most promising cancer biomarkers, lacks reliable quantitative methods other than enzyme-linked immunosorbent assay (ELISA). However, these methods suffer from limited sensitivity and a narrow linear range, often failing to achieve the low picograms per milliliter (pg / mL) sensitivity required for clinical applications. Therefore, developing a more sensitive and rapid MDK analysis method is crucial for early cancer diagnosis, prognostic assessment, and monitoring.

[0043] Based on the above situation, the present disclosure provides a reagent combination for a biosensor, which can be used to detect a variety of target proteins including MDK, and has the characteristics of high detection sensitivity, more convenient operation, and faster detection. The reagent combination includes NH2-MXene@AuNPs, biosynthetic quantum dots and platinum-coated gold nanorod probes.

[0044] MXene, a two-dimensional transition metal carbide / carbonitride material, is a novel two-dimensional nanomaterial derived from the MAX phase of layered ceramic materials by etching away the A element. It possesses a unique multilayered structure, excellent electrical conductivity, mechanical properties, and surface functionalization capabilities, and is environmentally friendly. It exhibits unique application potential in energy storage, catalysis, electromagnetic shielding, sensing, and biomedicine. Modification with functional groups or various nanomaterials can enhance MXene's performance in terms of electron transport, biocompatibility, and the exposure of more binding sites.

[0045] NH2-MXene material is an amino-modified MXene material. A large number of amino groups are introduced on the surface of the MXene material, making the surface smoother and the area larger.

[0046] AuNPs are gold nanoparticles. Metal nanoparticles are aggregates of metal atoms ranging in size from 1 to 100 nanometers, smaller than the wavelength of light. Their small size creates quantum confinement, where increasing or decreasing the number of metal atoms can significantly alter their structure, electronic, and optical properties. Therefore, unlike macroscopic metal materials, the size, morphology, and elemental distribution of metal nanoparticles determine their mechanical behavior, surface adsorption, transport, catalytic activity, and optoelectronic properties. These nanoparticles hold great promise for applications in the construction of biosensors, as well as in the study of electrochemical catalysis, optoelectronics, and physicochemical properties.

[0047] NH2-MXene@AuNPs is an amino-modified MXene material modified with gold nanoparticles. Anchoring gold nanoparticles on the amino-modified MXene material can effectively increase the conductivity of the sensing surface. When used in biosensors to detect and analyze analytes, it can effectively improve the sensitivity and speed of the biosensor.

[0048] Quantum dots are nanoscale semiconductor materials that have found widespread application in fields such as biosensing and solar cells. Biosynthesized quantum dots (BQDs) are synthesized using microbial biosynthesis. Traditionally, quantum dots are chemically synthesized, but microbial synthesis has been developed as an alternative. Microbial synthesis, due to the attachment of proteins and peptides to their surfaces, makes microbially synthesized quantum dots more biocompatible than chemically synthesized ones.

[0049] Platinum-coated gold nanorod probes (MF-Probes) can be used as multifunctional signal probes with excellent peroxidase-like activity. Their core-shell bimetallic nanomaterials provide excellent biocompatibility for antibody immobilization through Pt-N bonds. They also exhibit significant ability to improve electrochemical conductivity and have unique catalytic properties for H2O2 and TMB (3,3',5,5'-tetramethylbenzidine). Whether used in electrochemical biosensors or colorimetric biosensors, they can amplify signals and effectively improve detection sensitivity.

[0050] In some embodiments, the substrate material further comprises a first antibody against the target protein and bovine serum albumin.

[0051] In some embodiments, the platinum-coated gold nanorod probe is a probe modified with a secondary antibody against the target protein.

[0052] When the reagent combination disclosed in the present invention is used to detect the target protein, NH2-MXene@AuNPs, biosynthetic quantum dots, the first antibody of the target protein, and bovine serum albumin are first applied to the carrier in sequence to complete the preparation of the sensing substrate of the biosensor, and then the target protein and platinum-coated gold nanorod probes are added to achieve the determination of the target protein. The reagent combination for biosensors disclosed in the present invention can be used in electrochemical biosensors and can also be used in colorimetric biosensors. When applied to electrochemical biosensors, the carrier is a glassy carbon electrode, and when applied to colorimetric biosensors, the carrier is a well plate. It should be noted that those skilled in the art can apply the reagent combination disclosed in the present invention to other sensors as needed. They only need to replace the corresponding carrier as needed, and there is no specific limitation.

[0053] The NH2-MXene@AuNPs disclosed in the present invention have good electrical conductivity, so the electrical conductivity of the sensing surface can be ensured, and the large number of amino groups introduced can promote the connection with BQDs. BQDs are effectively fixed on the NH2-MXene@AuNPs material through their own carboxyl groups. Since BQDs are obtained by biosynthesis and have protein A, they can be accurately connected to the Fc fragment of the first antibody of the target protein, thereby playing a role in directional fixation and efficient recognition of the first antibody. Platinum-coated gold nanorod probes (MF-Probes) can be used as multifunctional signal probes with excellent peroxidase-like activity. Their core-shell bimetallic nanomaterials provide excellent biocompatibility for antibody fixation through Pt-N bonds, so they can effectively fix the second antibody of the target protein. The MF-Probe demonstrates remarkable ability to enhance electrochemical conductivity and possesses unique catalytic properties for H₂O₂ and TMB (3,3',5,5'-tetramethylbenzidine). Whether applied to electrochemical or colorimetric biosensors, it amplifies signals, effectively reducing false positive and false negative results during testing, thereby improving detection sensitivity and accuracy. Compared to existing biosensor reagents, the biosensor reagent combination provided in this disclosure can effectively improve detection sensitivity, accuracy, and speed, while effectively reducing detection difficulty. This is of great significance for the in-depth application of biosensors in medical clinical diagnosis.

[0054] The present disclosure also provides a biosensor for detecting a target protein, comprising a sensing substrate and a platinum-coated gold nanorod probe, wherein the sensing substrate is prepared from NH2-MXene@AuNPs and biosynthetic quantum dots.

[0055] The biosensor disclosed herein includes a sensing substrate and a platinum-coated gold nanorod probe. The sensing substrate is made of NH2-MXene@AuNPs and BQDs. When used to detect target proteins, NH2-MXene@AuNPs, as an underlying conductive material, has excellent electrochemical activity; BQDs are anchored to NH2-MXene@AuNPs, which can promote efficient recognition and directional fixation of antibodies; the platinum-coated gold nanorod probe can further amplify the electrical signal and provide excellent biocompatibility for antibody fixation. Through the cooperation of NH2-MXene@AuNPs, BQDs and MF-Probe, the sensitivity and accuracy of the biosensor can be effectively improved, and efficient detection of target proteins can be achieved.

[0056] The biosensor disclosed herein can be an electrochemical biosensor or a colorimetric biosensor. Regardless of the specific type of biosensor, it has the characteristics of high sensitivity, high accuracy, and convenience.

[0057] In some embodiments, the biosensor is an electrochemical biosensor; the sensing substrate of the electrochemical biosensor is prepared by the following method:

[0058] NH2-MXene@AuNPs, biosynthetic quantum dots, a first antibody of a target protein, and bovine serum albumin are sequentially applied to a glassy carbon electrode to obtain a sensing substrate of the electrochemical biosensor.

[0059] When the prepared biosensor is an electrochemical biosensor, the NH2-MXene@AuNPs solution is firstly coated onto the purified glassy carbon electrode. The nanostructured carbon electrode (N-MXene@AuNPs / GCE) was prepared by coating a BQD solution on the NH2-MXene@AuNPs / GCE to obtain a biosynthesized quantum dot-modified N-MXene@AuNPs / GCE. The BQD solution was then applied to the NH2-MXene@AuNPs / GCE to obtain a biosynthesized quantum dot-modified N-MXene@AuNPs / GCE. The first antibody (Ab1) of the target protein was applied to the surface of the BQDs / NH2-MXene@AuNPs / GCE for recognition and connection to obtain Ab1 / BQDs / NH2-MXene@AuNPs / GCE. Bovine serum albumin (BSA) was then used to debride the nonspecific binding sites and block the electrode surface to obtain the sensing substrate of the electrochemical biosensor (BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE).

[0060] In some embodiments, the biosensor is a colorimetric biosensor; the sensing substrate of the colorimetric biosensor is prepared by the following method:

[0061] NH2-MXene@AuNPs, biosynthetic quantum dots, the first antibody of the target protein, and bovine serum albumin are sequentially added dropwise to the well plate to obtain the sensing substrate of the colorimetric biosensor.

[0062] When the prepared biosensor is a colorimetric biosensor, the NH2-MXene@AuNPs solution is first drop-coated onto the well plate, and then the BQDs and the first antibody of the target protein are sequentially added to the well plate, and then BSA is added to clean the non-specific binding sites to obtain the final colorimetric biosensor sensing substrate. The electrochemical biosensor sensing substrate and the colorimetric biosensor sensing substrate prepared by the above method have excellent conductive properties, and under the action of BQDs, they can directionally identify and efficiently fix more first antibodies of the target protein, thereby improving the sensitivity and accuracy of the detection. When the electrochemical biosensor is used to detect the target protein, the electrochemical biosensor sensing substrate is placed in the target protein to be detected, and then the MF-Probe is added to complete the detection of the target protein. When the colorimetric biosensor is used to detect the target protein, the target protein is added drop-wise to the sensing substrate of the colorimetric biosensor, and then the MF-Probe is added to complete the detection of the target protein. The entire detection process is more efficient and convenient.

[0063] In some embodiments, prior to using the biosynthetic quantum dots to prepare biosensors, they must be activated. This activation process involves mixing the biosynthetic quantum dots with tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS), followed by stirring to activate the biosynthetic quantum dots. Whether preparing a sensing substrate for a colorimetric biosensor or an electrochemical biosensor, the biosynthetic quantum dots must be activated before use in the preparation of the corresponding biosensor sensing substrate.

[0064] In some embodiments, the platinum-coated gold nanorod probe is prepared by the following method:

[0065] Gold nanorods were mixed with CTAB solution, AgNO3 solution and ascorbic acid solution, stirred, and then HCl and H2PtCl6 solution were added to react to obtain platinum-coated gold nanorods;

[0066] Bovine serum albumin is added to the platinum-coated gold nanorod solution for incubation, and after the incubation is completed, a second antibody of the target protein is added for reaction to obtain the platinum-coated gold nanorod probe.

[0067] To synthesize MF-Probes, platinum-coated gold nanorods (Au@Pt NRs) must first be prepared, and then the Au@Pt NRs are used to prepare the MF-Probes. The synthesis process of Au@Pt NRs is as follows: First, the gold nanorods (AuNRs) are diluted in a three-necked flask. Then, a solution of cetyltrimethylammonium bromide (CTAB), a solution of silver nitrate (AgNO3), and a solution of ascorbic acid are added to the flask and stirred. Hydrochloric acid (HCl) and chloroplatinic acid (H2PtCl6) solutions are added to the flask and stirred continuously. After the reaction, the Au@Pt NRs are obtained. To obtain the MF-Probes, the Au@Pt NRs must be incubated. First, the Au@Pt NRs are added to BSA and incubated for a certain period of time. Then, a secondary antibody against the target protein is added and reacted for a certain period of time to obtain the MF-Probes. The platinum-coated gold nanorod probes prepared in this way can effectively improve the electrochemical conductivity and have unique catalytic properties for H2O2 and TMB. They can be successfully used in electrochemical biosensors and colorimetric biosensors to amplify signals, thereby further improving the detection sensitivity and accuracy of biosensors.

[0068] In some embodiments, the gold nanorods are prepared by the following method:

[0069] Mixing the HAuCl4 solution with the CTAB solution to obtain a first mixed solution;

[0070] Adding NaBH4 solution to the first mixed solution, stirring and allowing to stand to obtain a seed solution;

[0071] Completely dissolving CTAB and NaOL in deionized water to obtain a second mixed solution;

[0072] Adding AgNO3 solution, HAuCl4 solution and acrylic acid solution to the second mixed solution in sequence to obtain a growth solution;

[0073] The seed solution is added to the growth solution to react and generate the gold nanorods.

[0074] The specific synthesis steps for gold nanorods (AuNRs) are as follows: First, a solution of tetrachloroauric acid (HAuCl4) and a solution of cetyltrimethylammonium bromide (CTAB) are mixed in a flask to form a first mixed solution. Sodium borohydride (NaBH4) is then added dropwise to the flask (i.e., to the first mixed solution) while stirring, causing the solution's color to change from yellow to brown. The mixed solution in the flask is then allowed to passivate at room temperature, forming a seed solution. Subsequently, CTAB and sodium oleate (NaOL) are completely dissolved in a beaker containing deionized water to form a second mixed solution. After the second mixed solution is cooled, a solution of AgNO3 and a solution of HAuCl4 are added to the beaker (i.e., the second mixed solution) and stirred for a period of time, during which the color changes from yellow to colorless and transparent. Stirring is continued. Then, an acrylic acid solution (AA) is added and stirred vigorously to form a growth solution. The seed solution is added to the growth solution, shaken thoroughly, and allowed to stand for a period of time to react and form AuNRs.

[0075] In some embodiments, the NH2-MXene@AuNPs are prepared by the following method:

[0076] Ti2CTx-Mxene was added to an ethanol solution and subjected to ultrasonic treatment to obtain a Ti2CTx-Mxene-ethanol solution;

[0077] APTES solution was added to Ti2CTx-MXene-ethanol solution to obtain NH2-MXene;

[0078] Ultrasonic mixing of NH2-MXene and gold nanoparticles to obtain the NH2-MXene@AuNPs;

[0079] Wherein, the gold nanoparticles are prepared by the following method:

[0080] trisodium citrate was added to deionized water, heated to boiling, and then HAuCl4 solution was added to obtain a third mixed solution;

[0081] The trisodium citrate solution and the HAuCl4 solution were added to the third mixed solution in batches for multiple times to obtain the gold nanoparticles.

[0082] In order to obtain NH2-MXene@AuNPs, gold nanoparticles (AuNPs) and NH2-MXene need to be prepared separately.

[0083] The specific preparation method of NH2-MXene is as follows: titanium carbide-based MXene (Ti2CTx-MXene) is ultrasonically treated with an ethanol solution to obtain a Ti2CTx-MXene-ethanol solution; 3-aminopropyltriethoxysilane (APTES) is slowly added to the obtained Ti2CTx-MXene-ethanol solution, and the reaction is stirred to obtain a reaction product, which is centrifuged and washed with deionized water to remove unreacted APTES, and then dried in a vacuum dryer to obtain the final NH2-MXene.

[0084] The specific preparation method of AuNPs is as follows: trisodium citrate is added to deionized water, and then the solution is placed in a three-necked flask, stirred vigorously, and heated to boiling; then, HAuCl4 is slowly added to the three-necked flask, and the color of the solution changes from light yellow to pink within 10 minutes, indicating that gold seeds have been synthesized to obtain a third mixed solution; after the third mixed solution is cooled to 90°C, trisodium citrate solution and HAuCl4 solution are added dropwise, and the addition of trisodium citrate solution and HAuCl4 solution is repeated multiple times to react to obtain gold nanoparticles.

[0085] The preparation process of NH2-MXene@AuNPs is as follows: NH2-MXene is ultrasonically mixed with AuNPs to obtain NH2-MXene@AuNPs. The presence of -NH2 groups in NH2-MXene stably connects the AuNPs via Au-N covalent interactions, thus enabling the successful preparation of NH2-MXene@AuNPs.

[0086] In some embodiments, the biosynthetic quantum dots are prepared by the following method:

[0087] Staphylococcus aureus was activated in NB liquid culture medium, and then a portion of the bacterial solution was transferred to LB liquid culture medium for culture. CdCl2 and sterile water were added to the LB liquid culture medium and incubated to synthesize the biosynthetic quantum dots.

[0088] In order to synthesize biosynthetic quantum dots, it is necessary to first prepare nutrient broth (NB) liquid culture medium and lysogenic broth (LB) liquid culture medium. Beef extract, peptone and sodium chloride are dissolved in distilled water to obtain NB liquid culture medium. Yeast extract, peptone and sodium chloride are dissolved in distilled water to obtain LB liquid culture medium. Before using NB liquid culture medium and LB liquid culture medium, they need to be sterilized. Staphylococcus aureus is activated in NB liquid culture medium for a certain period of time, and then part of the bacterial solution is transferred to LB culture medium and cultured for a certain period of time to keep the cells in the stationary phase; sterile water containing cadmium chloride (CdCl2) is added to the culture medium and incubated for a certain period of time to allow intracellular biosynthesis to obtain BQDs.

[0089] In some embodiments, the target proteins include mid-term protein, alpha-fetoprotein, carcinoembryonic antigen, and prostate-specific antigen. The disclosed biosensor can be used not only to detect MDK, but also alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA). Its high sensitivity and rapid detection are of great significance for the early diagnosis, prognosis, and monitoring of cancer.

[0090] The present disclosure also provides an application of a biosensor in the detection of cancer markers, wherein a biosensor according to any of the above embodiments is used to detect the concentration of metaphase protein.

[0091] Hereinafter, MDK will be taken as an example to specifically illustrate the technical effects of the present disclosure.

[0092] Before detecting MDK, the corresponding sensors need to be prepared. In this example, an electrochemical biosensor and a colorimetric biosensor are prepared. Before preparing the corresponding biosensors, the reagent combination for the biosensor needs to be prepared. The complete preparation process is described in detail below.

[0093] (1) Synthesis of NH2-MXene@AuNPs

[0094] (① Preparation of NH2-MXene materials

[0095] 30 mg of Ti2CTx-MXene was ultrasonically treated with 15 mL of ethanol for 10 minutes to obtain a Ti2CTx-MXene-ethanol solution. 200 μL of APTES solution was then slowly added to the Ti2CTx-MXene-ethanol solution and stirred at 800 rpm for 24 hours to produce NH2-MXene. The reaction product was centrifuged at 4000 rpm, washed three times with deionized water to remove unreacted APTES, and then dried in a vacuum dryer at 50°C for 12 hours to obtain the final NH2-MXene product. The NH2-MXene was resuspended in deionized water to a concentration of 2 mg / mL and stored at 4°C until use.

[0096] ②Preparation of AuNPs

[0097] 51.76 mg of trisodium citrate was added to 80 mL of deionized water and mixed. The solution was then placed in a three-necked flask, stirred vigorously, and heated to boiling for 15 minutes. Subsequently, 533 μL of 25 mM HAuCl₄ was slowly added to the flask. The solution changed color from light yellow to pink within 10 minutes, indicating the synthesis of gold seeds. This yielded a third mixed solution. After cooling the third mixed solution to 90°C, 553 μL of 60 mM trisodium citrate solution and 533 μL of 25 mM HAuCl₄ solution were added dropwise. This addition of trisodium citrate solution and HAuCl₄ solution was repeated seven times to generate AuNPs. The reaction product was centrifuged, resuspended in deionized water, cooled to room temperature, and stored at 4°C until use.

[0098] (③NH 2- Preparation of MXene@AuNPs

[0099] 2 mL of NH2-MXene solution was ultrasonically mixed with 1 mL of AuNPs solution for 2 h to generate NH2-MXene@AuNPs. The reaction product was centrifuged at 3000 rpm and washed three times to obtain the final NH2-MXene@AuNPs. The NH2-MXene@AuNPs were stored at 4°C for later use.

[0100] (2) Synthesis of BQDs

[0101] Preparation of NB liquid medium: Dissolve 5 g of beef extract, 10 g of peptone, and 5 g of sodium chloride in 1 L of distilled water to obtain NB liquid medium.

[0102] Preparation of LB liquid medium: Dissolve 3 g of yeast extract, 10 g of peptone and 5 g of sodium chloride in 1 L of distilled water to obtain LB liquid medium.

[0103] NB liquid medium and LB liquid medium were sterilized. Staphylococcus aureus was activated in the sterilized NB liquid medium for 24 hours, and then 1 mL of the bacterial solution was transferred to a new sterilized LB medium and cultured for 12 hours (140 rpm, 37 ° C) to keep the cells in the stationary phase. Then, 500 μL of sterile water containing 11.4 mg of CdCl2 was added to the LB medium and incubated for 12 hours, and BQDs were biosynthesized intracellularly. The synthesized product was centrifuged at 4000 rpm, washed three times with Tris-HCl buffer (pH = 8, 0.1 M), and resuspended in the solvent. The optical density (OD) value was measured to determine the product concentration. The BQDs of the measured concentration were heated in a water bath at 65 ° C for 30 minutes to kill Staphylococcus aureus and terminate the reaction, and then stored at 4 ° C until use.

[0104] (3) Preparation of MF-Probe

[0105] (①Preparation of Au@Pt NRs

[0106] Step 1: Synthesis of AuNR

[0107] 5 mL of 0.5 mM HAuCl4 and 5 mL of 0.2 mM CTAB were mixed in a 20 mL flask to obtain a first mixed solution; 1 mL of 6 mM NaBH4 solution was added dropwise to the first mixed solution while stirring at 1200 rpm, and the color of the solution changed from yellow to brown; the mixed solution in the flask was then placed at room temperature for 30 minutes for passivation to obtain a seed solution. 9g of CTAB and 1.234g of NaOL were completely dissolved in a beaker containing 250mL of deionized water to obtain a second mixed solution. After the second mixed solution was cooled to 30°C, 18mL of 4mM AgNO3 solution was added to the beaker (i.e., the second mixed solution) and mixed thoroughly. 250mL of 1mM HAuCl4 solution was then added and the mixture was allowed to stand at 30°C for 15 minutes. The solution was then stirred at 700rpm for 90 minutes, maintaining the temperature. The color of the solution changed from yellow to colorless and transparent. After slow stirring at 400rpm for 15 minutes, 1.25mL of 0.064M AA was added and stirred vigorously for 30 seconds to obtain a growth solution. 400μL of the seed solution was then added to the growth solution, gently shaken to mix, and allowed to grow at 30°C for 12 hours to generate AuNRs. The resulting AuNRs were washed three times by centrifugation at 7000rpm in deionized water, resuspended in 20mL of deionized water, and stored in a refrigerator at 4°C until ready for use.

[0108] Step 2: Synthesis of Au@Pt NRs

[0109] In a three-necked flask, 250 μL of AuNR solution was diluted to 5 mL. Then, 10 mL of 0.05 M CTAB solution, 500 μL of 20 mM AgNO₃ solution, and 500 μL of 100 mM ascorbic acid solution were added to the flask and stirred at 70°C for 1 hour. Subsequently, 480 μL of 0.1 M HCl and 440 μL of 2 mM H₂PtCl₆ were added to the flask and stirred at 70°C for 4 hours to produce Au@Pt NRs. The reaction product was centrifuged at 8000 rpm for 10 min, washed twice, and resuspended in 1 mL of deionized water for use.

[0110] (② Preparation of MF-Probe

[0111] MF-Probes were prepared by adding 5 μL of 1% BSA solution to 200 μL of Au@Pt NRs solution and incubating at 4°C for 30 minutes. Then, 50 μL of a secondary antibody solution (10 μg / mL) against metaphase protein was added dropwise and reacted at 4°C for 12 hours. The MF-Probes were washed twice by centrifugation at 7000 rpm with pH 7.0 phosphate buffer (PBS) and resuspended in 200 μL of deionized water for later use.

[0112] (4) Preparation of sensing substrate for electrochemical biosensor

[0113] GCE purification: After being polished with nano-alumina powder and ultrasonically treated in deionized water and ethanol to remove surface adsorbed impurities, the GCE surface was dried with a nitrogen flow and stored at 4 °C.

[0114] Activation of biosynthesized quantum dots: 200 μL BQDs, 400 μL Tris-HCl, 0.2 M EDC, and 0.05 M NHS were mixed and stirred on a shaker at 26 °C for 30 min to achieve activation of biosynthesized quantum dots.

[0115] Preparation of sensing substrate:

[0116] 10 μL of NH2-MXene@AuNPs solution was drop-coated onto purified GCE and dried in air at room temperature to obtain NH2-MXene@AuNPs / GCE; 6 μL of activated BQDs solution was applied to NH2-MXene@AuNPs / GCE at 37°C for 30 minutes to obtain BQDs / NH2-MXene@AuNPs / GCE; then 10 μL of the first antibody (Ab1) solution (20 μg / mL) of mid-term protein was applied to the surface of BQDs / NH2-MXene@AuNPs / GCE for recognition and connection; 1% BSA solution was used to debride nonspecific binding sites and block the electrode surface to obtain the sensing substrate of the electrochemical biosensor (BSA / Ab1 / BQDs / NH2-Mxene@AuNPs / GCE).

[0117] (5) Preparation of Sensing Substrate for Colorimetric Biosensor

[0118] A 96-well plate was selected as the well plate for the colorimetric biobed sensor. 10 μL of NH2-MXene@AuNPs, 6 μL of activated BQDs, 10 μL of the first antibody (Ab1) solution (20 μg / mL) for midamin, and 1% BSA solution were sequentially added dropwise to the 96-well plate to obtain the sensing substrate of the colorimetric biosensor.

[0119] Below, some experiments are conducted to study the characteristics of NH2-MXene@AuNPs, BQDs, MF-Probe and biosensors to further demonstrate the technical effects of the present disclosure.

[0120] (1) Morphological characterization of Ti2CTx-MXene, NH2-MXene, AuNPs, and NH2-MXene@AuNPs

[0121] The morphologies of Ti2CTx-MXene, NH2-MXene, AuNPs and NH2-MXene@AuNPs were revealed by scanning electron microscope (SEM) and transmission electron microscope (TEM). Figure 1 shown.

[0122] from Figure 1 As can be seen in A, the layered Ti2CTx-MXene presents a loosely stacked accordion shape, while the NH2-MXene material modified by APTES, such as Figure 1 As shown in Figure 2B, its surface is smoother, the area is effectively expanded, and a large number of amino groups are introduced to promote the subsequent connection of BQDs.

[0123] from Figure 1 As can be seen from Figure C, well-dispersed single spherical nanoparticles are formed with a uniform diameter of 10 nanometers. Figure 2 ), which further confirms the single crystal form of AuNPs. Figure 1 As shown in Figure D, AuNPs are uniformly integrated on the surface of NH2-MXene, demonstrating the successful synthesis of NH2-MXene@AuNPs.

[0124] (2) Morphological characterization of BQDs

[0125] The morphology of BQDs was characterized by laser scanning confocal microscopy and TEM, and the size and elemental composition distribution of BQDs were analyzed. Figure 3 As shown. Figure 3 As shown in Figure A, small spherical particles with green fluorescence can be observed in Staphylococcus aureus under a laser scanning confocal microscope, confirming the synthesis of BQDs with good dispersion and relatively uniform size. Figure 3The unique morphology of Staphylococcus aureus can be seen in B and 3C, with a diameter of approximately 0.71 μm. The elemental composition of CdS4Se quantum dots isolated from Staphylococcus aureus was analyzed using X-ray photoelectron spectroscopy (XPS), as shown in Figure 3C. Figure 3 As shown in D, the presence of the three elements Cd, S and Se was confirmed in a ratio of approximately 1:4:2, further proving that quantum dots were synthesized inside Staphylococcus aureus and the synthesized quantum dot material was CdS4Se2.

[0126] (3) Characterization of the morphology and element distribution of AuNR and Au@Pt NRs

[0127] The morphology and element distribution of AuNR and Au@Pt NRs were characterized by TEM. Figure 4 As shown. Figure 4 As shown in Figures A and 4B , the gold nanorods are well dispersed, presenting a smooth and distinct rod-like structure with a uniform size of approximately 50 nm. Figure 4 The inset of B shows the clear lattice fringes of the gold nanorods, indicating that they are well crystalline with a lattice spacing of 0.23 nm, corresponding to the Au(gold) (111) crystal plane. These results indicate that the AuNRs synthesized by the seed growth method can be used as templates for the synthesis of Au@Pt NRs. Figure 4 As shown in Figures C and 4D, Pt (platinum) particles with a size of about 3-4 nm are evenly distributed on the surface of AuNR, forming a non-dense core-shell structure according to the Volmer-Weber growth model. Figure 4 In the inset of D, clear lattice fringes can be observed, and the lattice spacing is determined to be 0.241 nm, corresponding to the Pt(111) crystal plane.

[0128] The element distribution of Au@Pt NRs was characterized by EDX, as shown in Figure 2. Figure 5 As shown, it shows that the nanocomposite material is composed of Au and Pt elements.

[0129] (4) Study on the electrical conductivity of GCE, NH2-MXene / GCE, and NH2-MXene@AuNPs / GCE

[0130] Containing 10mM [Fe(CN)6] 3- The conductivity properties of GCE, NH2-MXene / GCE, and NH2-MXene@AuNPs / GCE were evaluated by cyclic voltammetry (CV) with a voltage range of -0.2 V to 0.6 V and a scan rate of 50 mV / s. Figure 6As shown in A. Since NH2-MXene has excellent electrical conductivity, it can effectively enhance the current signal after combining with GCE. Figure 6 As can be seen in Figure A, NH2-MXene / GCE (curve b) exhibits a significantly enhanced current signal compared to bare GCE (curve a). NH2-MXene@AuNPs / GCE (curve c) exhibits the highest current response, with an Ipa value of 98.65 μA. This indicates that the binding of NH2-MXene to gold nanoparticles further enhances the current signal, making it more sensitive when used in biosensors to detect target proteins.

[0131] The electroactive surface area of ​​different materials can be quantitatively estimated using the Randles-Sevcik equation (Li et al. 2021):

[0132] I pa =2.69×10 5 n 2 / 3 A eff D 1 / 2 v 1 / 2 C0

[0133] Among them I pa represents the anode peak current, n represents the number of transferred electrons, A eff Represents the electroactive surface area (cm 2 ), D is [Fe(CN)6] 3- The diffusion coefficient of the redox probe (equal to 1) is (6.70±0.02)×10 -6 cm 2 / s, v is the scanning rate, C0 is the concentration of the redox probe (10 mmol / cm -3 ). According to the above formula, the surface area of ​​NH2-MXene@AuNPs / GCE is calculated to be 6.4×10 -2 cm 2 , which are 1.58 and 2.02 times those of NH2-MXene / GCE and bare GCE, respectively. The combined modification of gold nanoparticles and -NH2 groups effectively increases the active surface area, making NH2-MXene@AuNPs have excellent electrochemical activity, which can be used as a bottom conductive material in biosensors.

[0134] (5) Study on the antibody capture ability of BQDs

[0135] In order to study the antibody capture ability of BQDs, electrodes modified with NH2-MXene@AuNPs and BQDs / NH2-MXene@AuNPs composite materials, namely NH2-MXene@AuNPs / GCE and BQDs / NH2-MXene@AuNPs / GCE, were prepared. Under the same conditions, the electrodes modified with NH2-MXene@AuNPs and BQDs / NH2-MXene@AuNPs were incubated with 20 μg / mL MDK antibody at 37°C for 1 hour. After incubation, the electrodes were incubated in a solution containing 10 mM [Fe(CN)6] 3- The peak current difference (ΔI / μA) of the two composite materials before and after binding to the MDK antibody was measured using differential pulse voltammetry (DPV) in a 0.1 M KCl solution. The results are shown in FIG. Figure 6 As shown in Figure B. After incubation with MDK antibodies, the ΔI on NH2-MXene@AuNPs / GCE (curves b, d) is 32.70 μA, while the ΔI on BQDs / NH2-MXene@AuNPs / GCE (curves a, c) drops to 50.45 μA. The current decreases significantly, and the resistance increases significantly, indicating that more MDK antibodies are immobilized on BQDs / NH2-MXene@AuNPs / GCE after antibody incubation. This further proves that BQDs can promote the immobilization of antibodies and improve the capture ability of antibodies. When applied to biosensors, they can effectively improve the biosensor's ability to capture antibodies, thereby improving detection sensitivity and speed.

[0136] (6) Study on the electrochemical performance of MF-Probe

[0137] The electrochemical performance of MF-Probe was studied using DPV. 0.1 ng / mL MDK was detected using an electrochemical biosensor in a PBS buffer solution containing 5 mM H2O2. The control group used only the electrochemical biosensor substrate to detect MDK without adding MF-Probe. The results are shown in Figure 2. Figure 6 As shown in C. Figure 6 As can be seen in Figure C, in the absence of the MF-Probe, the current is relatively small, only 9.66 μA. In the presence of the MF-Probe, the attractive and catalytic properties of the Au@Pt NRs lead to rapid H₂O₂ redox reactions, increasing the current signal to 18.46 μA. This represents a 1.91-fold increase compared to the counterpart without the MF-Probe, effectively demonstrating the excellent signal amplification capabilities of the MF-Probe. Therefore, the biosensor fabricated using it can effectively amplify the signal, improving detection sensitivity and accuracy.

[0138] (7) Potential applications of MF-Probe in colorimetric detection

[0139] In order to further study the potential application of MF-Probe in colorimetric biosensor detection and eliminate the influence of substrate materials on the colorimetric mode, a two-component TMB colorimetric substrate (containing TMB and H2O2) was used. Figure 6 As shown in Figure D, the control (deionized water) and NH2-MXene@AuNPs exhibit minimal absorption peaks, while the MF-Probe exhibits a distinct absorption peak at 650nm. These results demonstrate that Au@Pt NRs possess peroxidase-like activity, oxidizing the H2O2 in the TMB two-component reagent and causing a color change in TMB. Therefore, the MF-Probe can also serve as a signal probe for MDK detection in colorimetric mode, and the colorimetric biosensor prepared using it exhibits high sensitivity and accuracy.

[0140] (8) Research on the manufacturing process of electrochemical biosensors

[0141] Electrochemical impedance spectroscopy (EIS) and DPV were used to verify the electrochemical biosensor fabrication process. The results were as follows: Figure 6 As shown in Figures 6E and 6F, the Rct value of the electrode modified with NH2-MXene@AuNPs and BQDs (curves b and c) is significantly lower than that of the unmodified bare GCE (curve a), indicating that NH2-MXene@AuNPs and BQDs can effectively promote the electron transfer rate on the electrode surface; after modification with the first antibody of MDK (curve d), BSA (curve e), MDK (curve f) and MF-Probe (curve g), higher impedance can be observed. The gradual increase in resistance indicates that the non-conductive bioactive substances have been successfully modified layer by layer, indicating that the antigen and antibody are successfully attached to the electrode, which further indicates that the electrochemical biosensor has been successfully prepared and can be successfully applied to the detection of MDK.

[0142] (9) Research on the application of biosensors in MDK detection

[0143] ① Detection of MDK concentration using electrochemical biosensor

[0144] Prepare MDK solutions with concentrations of 5fg / mL, 10fg / mL, 50fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, and 10ng / mL, place the sensing substrate of the electrochemical biosensor (BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE) in a container containing the MDK solutions at the above concentrations, and then add MF-Probe to obtain the final configuration:

[0145] MF-Probe / MDK / BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE. During each modification process, the sensing substrate of the electrochemical biosensor was gradually washed with 0.1M PBS buffer solution (pH 7.4) to remove unattached molecules on the electrode surface at 4°C until the next electrochemical measurement. The test results are shown in Figure 7 A. At the same time, the detection results of the electrochemical biosensor were calibrated to obtain a calibration curve, where the error bar = SD (n = 3), as shown in Figure 7 As shown in B.

[0146] ② Detection of MDK concentration using colorimetric biosensor

[0147] Prepare MDK solutions with concentrations of 5pg / mL, 10pg / mL, 100fg / mL, 1ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 50ng / mL, and 100ng / mL. Add the MDK solutions of the above concentrations dropwise to the sensing substrate of the colorimetric biosensor, then add MF-Probe and 150μL TMB two-component colorimetric solution. Incubate at 37°C in the dark for 15 minutes, and add 75μL of 5mM H2SO4 solution to terminate the reaction. Use a multiplate reader to scan the sample at 1nm intervals within the wavelength range of 300-600nm to obtain the absorbance spectrum of each well. The results are shown in Figure 2. Figure 7 C; the measured absorbance was calibrated to obtain the corresponding calibration curve, where the error bar = SD (n = 3), and the results are shown in FIG. Figure 7 D. In addition, photos were taken with a smartphone within 15 minutes, and the results were as follows Figure 7 E, and read using ImageJ Figure 7 The RGB values ​​of the colors in E were used to obtain a linear relationship between the RGB values ​​and the MDK concentration, where the error bars = SD (n = 3), as shown in Figure 7 As shown in F.

[0148] exist Figure 7In Figure A, it can be seen that the electrochemical signal intensity increases with the increase of MDK concentration, and the relative peak current ΔI (ΔI = I-I0, where I represents the current response corresponding to different concentrations of midkine and I0 represents the current response at 0 fg / mL) has a good linear relationship with the logarithmic value of MDK in the range of 5 fg / mL to 10 ng / mL (e.g. Figure 7 B), the linear regression equation is ΔI=1.688×lg c +9.758(R 2 =0.994). The limit of detection (LOD) can be calculated as LOD = KS_D / m, where K is the confidence constant, typically K = 3, SD is the relative standard deviation of 10 blank values, and m is the slope of the calibration curve within the concentration range. The calculated limit of detection is 1.620 fg / mL (signal-to-noise ratio of 3).

[0149] Due to the special properties of MF-Probe, MDK can also be detected by UV-visible spectroscopy (such as Figure 7 C, D) and mobile phones (such as Figure 7 E, F) is realized. Figure 7 As shown in C and 7E, the absorption peak at 450nm rises sharply with the increase of MDK, and the grayscale value reading of RGB color also increases significantly. Figure 7 D) It can be seen that the sensor exhibits two linear ranges. In the MDK concentration range of 5 pg / mL to 5 ng / mL, the linear regression equation is I = 0.300 × lg c +1.239(R 2 =0.996), in the range of 5ng / mL to 100ng / mL, the linear regression equation is I = 1.213×lg c +0.629(R 2 =0.992), the LOD of the colorimetric determination was 2.295 pg / mL; the linear regression equation obtained in Image 7F was y = 0.301 × lg c +0.037(R 2 =0.993), with a wide linear range of 10 pg / mL to 100 ng / mL and a low LOD of 1.930 pg / mL.

[0150] It can be seen that the biosensor proposed in the present disclosure can not only select a mode according to specific detection requirements, but also form a self-calibration mechanism through signals between different modes, making MDK detection more convenient, rapid and accurate.

[0151] (10) Research on the application of biosensors in clinical diagnosis of cancer

[0152] Sample Description: Samples for this test were collected from Tianjin Medical University and the Tianjin Medical University Cancer Research Institute and Hospital. Samples included both healthy donors and donors from thyroid cancer patients. All test samples underwent specific pretreatment after collection. This included gently inverting the collected blood sample 10 times and then placing it at 37°C for 2 hours until separation. Serum was then obtained from the separated blood sample by centrifugation at 3000 rpm for 10 minutes and stored at -80°C. To minimize the impact of other impurities in the serum, the serum was diluted 32-fold before use as the final test sample.

[0153] The electrochemical biosensor, colorimetric biosensor and ELISA device disclosed in the present invention were used to detect the MDK concentration in the serum of healthy donors and thyroid cancer patient donors. The results are as follows: Figure 8 As shown. Figure 8 As can be seen in Figure A, the detection results of the electrochemical biosensor and the colorimetric biosensor are consistent with the signal changes of the commercial ELISA detection, and for the same sample, the signal intensity of the biosensor proposed in the present disclosure is higher than its ELISA counterpart, which effectively demonstrates that the biosensor proposed in the present disclosure has higher sensitivity. Figure 8 As shown in B, the MDK level in thyroid cancer patients was much higher than that in healthy volunteers. Using t-test, it was found that the average MDK level between thyroid cancer patients and healthy donors was significantly different (P<0.0001), indicating that MDK is an ideal biomarker for clinical diagnosis (such as Figure 8 C). In addition, the receiver operating characteristic (ROC) curve showed that our proposed biosensor achieved high-accuracy cancer diagnosis (AUC = 1) and successfully distinguished thyroid cancer patients from healthy individuals (e.g. Figure 8 D). This effectively demonstrates that the biosensor disclosed herein can be used for clinical diagnosis of thyroid cancer, and also indirectly demonstrates the potential of midkine as a cancer diagnostic biomarker.

[0154] The present disclosure provides a reagent combination for a biosensor, a biosensor, and its application in cancer marker detection, wherein AuNPs are covalently anchored to an aminated MXene to ensure the conductivity of the sensing surface. Since AuNPs can covalently bind to the -SH and -NH2 groups in the first antibody of the target protein through Au-S or Au-N bonds, a certain amount of the first antibody of the target protein can be randomly fixed. At the same time, compared with the random binding of AuNPs to the first antibody of the target protein in NH2-MXene@AuNPs, the introduced BQDs have a high density of protein A and carboxyl groups on their surface. The carboxyl groups can tightly connect the BQDs to the NH2-MXene@AuNPs through amide bonds, and the protein A can specifically bind to the Fc region of the first antibody of the target protein, thereby increasing the capture of the first antibody and more exposure of the active binding site, thereby effectively improving the detection sensitivity, accuracy and speed.

[0155] On this basis, the platinum-coated gold nanorod probe (MF-Probe) has the function of amplifying signals and is used as a multifunctional signal probe with excellent peroxidase-like activity. The core-shell bimetallic nanomaterial of the platinum-coated gold nanorod provides excellent biocompatibility for antibody fixation through Pt-N bonds, exhibits significant ability in improving electrochemical conductivity, and has unique catalytic properties for H2O2 and TMB. When using electrochemical and colorimetric methods to determine midkine levels, it can minimize false positive and false negative results, further improving detection sensitivity and accuracy.

[0156] The disclosed biosensor achieves a wide detection range from 5 fg / mL to 100 ng / mL, with a limit of detection (LOD) of 1.620 fg / mL. It can be detected using an electrochemical workstation, microplate reader, or even a mobile phone. Compared with existing ELISAs, it not only has a lower LOD but is also more sensitive to trace MDK, enabling high-precision differentiation of subtle changes in MDK levels in the serum of healthy individuals and thyroid cancer patients. The disclosed biosensor can be used to assess trace MDK levels in complex matrices, contributing to in-depth research into the role of MDK in cancer screening and prognostic assessment, and is of great significance for the clinical diagnosis and prognostic assessment of cancer.

[0157] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present application (including the claims) is limited to these examples. Within the scope of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0158] In addition, when details are set forth to describe exemplary embodiments of the present application, it will be apparent to those skilled in the art that the present application embodiments can be implemented without these details or with variations in these details. Therefore, these descriptions should be considered illustrative rather than restrictive.

[0159] For simplicity of description and discussion, and in order not to make the embodiment of the application difficult to understand, the known power supply / ground connection with other components may or may not be shown in the accompanying drawings provided. In addition, the device can be shown in the form of a block diagram to avoid making the embodiment of the application difficult to understand, and this also takes into account the following fact, that is, the details of the embodiment of these block diagram devices are highly dependent on the platform to be implemented in the embodiment of the application (that is, these details should be fully within the scope of understanding of those skilled in the art). When specific details are set forth to describe the exemplary embodiments of the application, it will be apparent to those skilled in the art that the embodiment of the application can be implemented without these specific details or when these specific details are changed. Therefore, these descriptions should be considered to be illustrative rather than restrictive.

[0160] Although the present application has been described in conjunction with specific embodiments of the present application, many substitutions, modifications, and variations of these embodiments will be apparent to those skilled in the art based on the foregoing description. The present application embodiments are intended to encompass all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application embodiments should be included within the scope of protection of the present application.

Claims

1. A biosensor, characterized in that: Used to detect target proteins, comprising a sensing substrate and a platinum-coated gold nanorod probe, wherein the biosensor is an electrochemical biosensor or a colorimetric biosensor; The sensing substrate of the electrochemical biosensor is prepared by the following method: NH2-MXene@AuNPs, biosynthetic quantum dots, a first antibody of a target protein, and bovine serum albumin are sequentially applied to a glassy carbon electrode to obtain a sensing substrate of the electrochemical biosensor; The sensing substrate of the colorimetric biosensor is prepared by the following method: NH2-MXene@AuNPs, biosynthetic quantum dots, the first antibody of the target protein, and bovine serum albumin are sequentially added dropwise to the well plate to obtain the sensing substrate of the colorimetric biosensor; The platinum-coated gold nanorod probe is prepared by the following method: Gold nanorods were mixed with CTAB solution, AgNO3 solution and ascorbic acid solution, stirred, and then HCl and H2PtCl6 solution were added to react to obtain platinum-coated gold nanorods; Adding bovine serum albumin to a platinum-coated gold nanorod solution for incubation, and after the incubation is complete, adding a second antibody to the target protein to react, thereby obtaining the platinum-coated gold nanorod probe; The gold nanorods are prepared by the following method: Mixing the HAuCl4 solution with the CTAB solution to obtain a first mixed solution; Adding NaBH4 solution to the first mixed solution, stirring and allowing to stand to obtain a seed solution; Completely dissolving CTAB and NaOL in deionized water to obtain a second mixed solution; Adding AgNO3 solution, HAuCl4 solution and acrylic acid solution to the second mixed solution in sequence to obtain a growth solution; The seed solution is added to the growth solution to react and generate the gold nanorods.

2. The biosensor according to claim 1, wherein The NH2-MXene@AuNPs were prepared by the following method: Ti2CTx-Mxene was added to an ethanol solution and subjected to ultrasonic treatment to obtain a Ti2CTx-Mxene-ethanol solution; APTES solution was added to Ti2CTx-MXene-ethanol solution to obtain NH2-MXene; Ultrasonic mixing of NH2-MXene and gold nanoparticles to obtain the NH2-MXene@AuNPs; Wherein, the gold nanoparticles are prepared by the following method: trisodium citrate was added to deionized water, heated to boiling, and then HAuCl4 solution was added to obtain a third mixed solution; The trisodium citrate solution and the HAuCl4 solution were added to the third mixed solution in batches for multiple times to obtain the gold nanoparticles.

3. The biosensor according to claim 1, wherein The biosynthetic quantum dots are prepared by the following method: Staphylococcus aureus was activated in NB liquid culture medium, and then a portion of the bacterial solution was transferred to LB liquid culture medium for culture. CdCl2 and sterile water were added to the LB liquid culture medium and incubated to synthesize the biosynthetic quantum dots.

4. The biosensor according to claim 1, wherein The target proteins include metaphase protein, alpha-fetoprotein, carcinoembryonic antigen and prostate specific antigen.

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